Optically active agents have historically been used in the research setting for monitoring electrophysiology. As one example, voltage-sensitive and calcium-sensitive fluorescent dyes can be used to monitor cardiac electrical activity and associated contractile function. However, these techniques have not been applied in a clinical setting, nor usefully in living large animal models.
Further, optical imaging of voltage and calcium dyes requires, among other things, light sensors (e.g., CCD, CMOS, etc.) capable of measuring small intensity fluctuations on a large baseline signal at a high frame-rate, adequate illumination in one or more wavelength ranges, and/or filtering of excitation and/or illumination light.
A wide range of such dyes exist for use in research and are widely used in explanted hearts (e.g., Langendorff preparations) and in small animal models. These include conventional dyes such as Di-4-ANEPPS as well as newer generation ratiometric dyes and near IR emission dyes. Relatedly, techniques are even emerging to genetically encode proteins with similar optical activity.
Dyes of this type are typically lipophilic (to varying degrees) and are rapidly taken up into the cell wall as they pass through a capillary bed. Experimentally, loading of these dyes is most often performed in a substantially blood free environment, e.g., where tissue blood perfusion is either briefly, or in a sustained manner replaced by an alternative fluid that is largely devoid of cells. A variety of such preparations exist for Langendorff models, particularly those deriving from small mammals. In a live small animal model, blood may be temporarily excluded from the vessels and/or tissue, for example, by infusion of large volumes of saline. Dye may be put into solution with or without amphipathic agents (e.g., DMSO, PEG, etc.) and loading may take place in this environment.
When blood is present, lipophilic dyes may be rapidly consumed by red blood cells, white blood cells, platelets, etc. before they are able to reach the capillary bed and be absorbed by cardiomyocytes, etc. Additionally, when flow rates are high, dye may be rapidly diluted, further decreasing the effective concentration that is delivered to the capillary bed.
Due to the complexity of loading dyes in-vivo and the optical systems required for imaging, minimally invasive techniques and tools for in-vivo optical mapping have not been developed.
Therefore, improved methods for loading dyes in blood perfused tissue and imaging tissues loaded with such dyes would be useful.